This application is directed to a process for preparing phosphorothioate oligonucleotides and to intermediates used in that process. Nucleoside precursors are phosphitylated with S-(alkaryl or aryl) alkyl phosphorothioate diester salts and then reacted in solution phase to build phosphorothioate oligonucleotides.
It is well known that most of the bodily states in mammals including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused upon interactions with such proteins in an effort to moderate their disease causing or disease potentiating functions. Recently, however, antisense methodology has been introduced to moderate the actual production of such proteins by interactions with messenger RNA (mRNA) or other intracellular RNA's that direct protein synthesis or with DNA's that direct the production of RNA. It is a general object of such therapeutic approaches to interfere with or otherwise modulate the expression of genes associated with undesired protein formation.
Antisense methodology is the complementary hybridization of relatively short oligonucleotides to single-stranded RNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization involves sequence specific hydrogen bonding via Watson-Crick base pairs of the heterocyclic bases of oligonucleotides to RNA or DNA. Such base pairs are said to be complementary to one another.
Events that provide for the disruption of the nucleic acid function, as discussed by Cohen in Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Inc., Boca Raton, Fla. (1989), are thought to include at least two types. The first is hybridization arrest. This denotes a terminating event in which an oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides such as disclosed by Miller, P. S. and Ts'O, P. O. P. (1987) Anti-Cancer Drug Design, 2:117-128, and .alpha.-anomer oligonucleotides are the two most extensively studied antisense agents that are thought to disrupt nucleic acid function by hybridization arrest.
In determining the extent of hybridization arrest of an oligonucleotide, the relative ability of an oligonucleotide to bind to complementary nucleic acids may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (T.sub.m), a characteristic physical property of double helixes, denotes the temperature in degrees centigrade at which 50% helical (hybridized) versus coil (unhybridized) forms are present. T.sub.m is measured by using the UV spectrum to determine the formation and breakdown (melting) of hybridization. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher T.sub.m. The higher the T.sub.m, the greater the strength of the binding of the strands. Non-Watson-Crick base pairing, i.e. base mismatch, has a strong destabilizing effect seen as a decrease in T.sub.m.
A second type of terminating event for antisense oligonucleotides involves the enzymatic cleavage of the targeted RNA by intracellular RNase H. The mechanism of RNase H cleavages requires the hybridization of a 2'-deoxyribofuranosyl oligonucleotide to a targeted RNA. The resulting DNA-RNA duplex activates the RNase H enzyme. The activated enzyme cleaves the RNA strand. Cleavage of the RNA strand destroys the normal function of the RNA. Phosphorothioate oligonucleotides are one prominent example of antisense agents that operate by this type of antisense terminating event.
The current method of choice for the preparation of phosphorothioate oligonucleotides is via solid-phase synthesis wherein an oligonucleotide is prepared on a polymer support. Solid-phase synthesis relies on sequential addition of nucleotides to one end of a growing oligonucleotide. Typically, a first nucleoside is attached to an appropriate glass bead support and nucleotide phosphoramidites are added stepwise to elongate the growing oligonucleotide. The nucleotide phosphoramidites are reacted with the growing oligonucleotide using the principles of a "fluidized bed" for mixing of the reagents. The known silica supports suitable for anchoring the oligonucleotide are very fragile and thus can not be exposed to aggressive mixing. Brill, W. K.-D., Tang, J.-Y., Ma, Y.-Y. and Caruthers, M. H. (1989) J. Am. Chem. Soc., 111: 2321 disclosed a procedure wherein an aryl mercaptan is substituted for the nucleotide phosphoramidite to prepare phosphorodithioate oligonucleotides on glass supports.
In these and other solid-phase procedures the oligonucleotide is synthesized as an elongating strand. However, the number of individual strands that can be anchored to a unit surface area of the support is limited. Also, the activated nucleotides that are added to the growing oligonucleotide are relatively expensive and must be used in stoichiometric excess.
While presently-utilized solid-phase syntheses are very useful for preparing small quantities of oligonucleotide, i.e., up to about 0.4 mole per synthetic run, they typically are not amenable to the preparation of the larger quantities of oligonucleotides necessary for biophysical studies, pre-clinical and clinical trials and commercial production. A general review of solid-phase verse solution-phase oligonucleotide synthesis is given in the background section of U.S. Pat. No. 4,517,338, entitled Multiple Reactor System And Method For Polynucleotide Synthesis, to Urdea, et al.
Solution-phase synthetic oligonucleotide techniques should be useful for large scale preparation. One such solution phase preparation utilizes phosphorus triesters. As I reported [Yau, E. K., Ma, Y.-S. and Caruthers (1990) Tetrahedron Letters, 31:1953], the triester oligonucleotide approach can be utilized to prepare thymidine dinucleoside and thymidine dinucleotide phosphorodithioates. The phosphorylated thymidine nucleoside intermediates utilized in this approach were obtained by treatment of commercially available 5'-O-dimethoxytritylthymidine-3'-[(.beta.-cyanoethyl)-N,N-diisopropyl]-pho sphoramidite first with either 4-chloro or 2,4-dichlorobenzylmercaptan and tetrazole and then a saturated sulfur solution. The resulting phosphorodithioate nucleotide was then reacted via the triester synthesis method with a further thymidine nucleoside having a free 5'-hydroxyl.
Brill, W. K.-D., Nielsen, J. and Caruthers, M. H. (1991) J. Am. Chem. Soc., 113:3972, recently disclosed that treatment of a phosphoramidite such as N,N-diisopropyl phosphoramidite with a mercaptan such as 4-chloro or 2,4-dichlorobenzylmercaptan in the presence of tetrazole yields a derivative suitable for preparation of a phosphorodithioate as a major product and a derivative suitable for preparation of a phosphorothioate as a minor product. Substituting 4-chloro or 2,4-dichlorobenzylmercaptan for N,N-diisopropyl phosphoramidite therefore would be expected to result in low yields of phosphorothioate oligonucleotides.
Thus, although the utility of phosphorothioate oligonucleotides in antisense methodology has been recognized, the art suggests no large scale techniques for their preparation. Accordingly, there remains a long-felt need for such methods and for intermediates useful in such methods.